U.S. patent application number 12/863368 was filed with the patent office on 2011-02-24 for composition comprising the extracts of lindera obtusiloba for prevention and treatment of cardiovascular diseases.
This patent application is currently assigned to YANG JI CHEMICAL CO., LTD.. Invention is credited to Woon-Sun Choi, Jong-Hoon Kim, Jung-Ok Lee, Jee Woong Lim, Min-Ho Oak, Chung Keun Rhee, Jung-Duk Sohn.
Application Number | 20110045111 12/863368 |
Document ID | / |
Family ID | 40885750 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110045111 |
Kind Code |
A1 |
Oak; Min-Ho ; et
al. |
February 24, 2011 |
COMPOSITION COMPRISING THE EXTRACTS OF LINDERA OBTUSILOBA FOR
PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES
Abstract
The present invention relates to a composition for treatment and
prevention of cardiovascular diseases, containing an extract of
Lindera obtusiloba. More particularly, the extract of Lindera
obtusiloba strongly inhibits NAD(P)H oxidases, which are the main
cause of vascular diseases, and at the same time, regulates
contraction and relaxation of vascular smooth muscles to manifest a
potent vasorelaxant effect, thereby improving the blood pressure
regulation and vascular endothelial dysfunction. Therefore, a
composition containing this as an active ingredient can be usefully
utilized as a pharmaceutical product or health functional food
product for the prevention and treatment of cardiovascular
diseases.
Inventors: |
Oak; Min-Ho; (Gunpo, KR)
; Lee; Jung-Ok; (Ansan, KR) ; Choi; Woon-Sun;
(Ulsan, KR) ; Sohn; Jung-Duk; (Anyang, KR)
; Kim; Jong-Hoon; (Anyang, KR) ; Lim; Jee
Woong; (Seongnam, KR) ; Rhee; Chung Keun;
(Seoul, KR) |
Correspondence
Address: |
CANTOR COLBURN LLP
20 Church Street, 22nd Floor
Hartford
CT
06103
US
|
Assignee: |
YANG JI CHEMICAL CO., LTD.
Ansan
KR
HAN WHA PHARMA CO., LTD.
Chuncheon
KR
|
Family ID: |
40885750 |
Appl. No.: |
12/863368 |
Filed: |
December 17, 2008 |
PCT Filed: |
December 17, 2008 |
PCT NO: |
PCT/KR08/07484 |
371 Date: |
October 18, 2010 |
Current U.S.
Class: |
424/774 ;
424/725; 424/779 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
9/04 20180101; A61P 9/10 20180101; A61P 9/00 20180101; A61P 3/06
20180101; A61P 9/02 20180101; A61P 43/00 20180101; A23L 33/105
20160801; A23V 2002/00 20130101; A61P 9/06 20180101; A61P 3/00
20180101; A61P 9/08 20180101; A61K 36/54 20130101; A61P 25/28
20180101; A61K 36/54 20130101; A61K 2300/00 20130101; A23V 2002/00
20130101; A23V 2200/326 20130101; A23V 2250/21 20130101 |
Class at
Publication: |
424/774 ;
424/725; 424/779 |
International
Class: |
A61K 36/54 20060101
A61K036/54; A61P 9/00 20060101 A61P009/00; A61P 3/06 20060101
A61P003/06; A61P 9/10 20060101 A61P009/10; A61P 3/00 20060101
A61P003/00; A61P 9/12 20060101 A61P009/12; A61P 9/06 20060101
A61P009/06; A61P 9/02 20060101 A61P009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 18, 2008 |
KR |
10-2008-0005672 |
Claims
1. A composition for prevention and treatment of a cardiovascular
disease, comprising an extract of Lindera obtusiloba as an active
ingredient.
2. The composition for prevention and treatment of a cardiovascular
disease according to claim 1, wherein the Lindera obtusiloba is
selected from the group consisting of twigs, leaves and mixtures
thereof.
3. The composition for prevention and treatment of a cardiovascular
disease according to claim 1, wherein the extract of Lindera
obtusiloba is extracted with a solvent selected from the group
consisting of water, a C.sub.1-C.sub.5 lower alcohol, and a mixture
thereof.
4. The composition for prevention and treatment of a cardiovascular
disease according to claim 3, wherein the extract of Lindera
obtusiloba is extracted with a 30 to 95 wt % aqueous solution of
ethanol.
5. The composition for prevention and treatment of a cardiovascular
disease according to claim 3, wherein the extract of Lindera
obtusiloba is extracted with a solvent selected from the group
consisting of water, a C.sub.1-C.sub.3 lower alcohol and a mixture
thereof, and the resultant is re-extracted with butanol.
6. The composition for prevention and treatment of a cardiovascular
disease according to claim 1, wherein the extract of Lindera
obtusiloba has an NAD(P)H oxidase inhibitory activity.
7. The composition for prevention and treatment of a cardiovascular
disease according to claim 1, wherein the extract of Lindera
obtusiloba has a vasorelaxant effect.
8. The composition for prevention and treatment of cardiovascular
diseases according to claim 1, wherein the extract of Lindera
obtusiloba has an effect of increasing the activity of endothelial
type nitric oxide synthase.
9. The composition for prevention and treatment of a cardiovascular
disease according to any one of claims 1 to 8, wherein the
cardiovascular disease is selected from the group consisting of
congestive heart diseases, coronary artery diseases (heart attack),
ischemic heart diseases (myocardial ischemia), hyperlipidemia,
arteriosclerosis, hypertension, hypotension, arrhythmia, cardiac
failure, vascular restenosis, cerebrovascular diseases (cerebral
stroke, dementia), peripheral vascular diseases and metabolic
diseases.
10. A health functional food product for prevention of a
cardiovascular disease, comprising an extract of Lindera obtusiloba
as an active ingredient.
11. A health functional food product for prevention of a
cardiovascular disease according to claim 10, wherein the Lindera
obtusiloba is selected from the group consisting of twigs, leaves
and mixtures thereof.
12. A health functional food product for prevention of a
cardiovascular disease according to claim 10, wherein the extract
of Lindera obtusiloba is extracted with a solvent selected from the
group consisting of water, a C.sub.1-C.sub.5 lower alcohol, and a
mixture thereof.
13. A health functional food product for prevention of a
cardiovascular disease according to claim 10, wherein the extract
of Lindera obtusiloba is extracted with a solvent selected from the
group consisting of water, a C.sub.1-C.sub.3 lower alcohol and a
mixture thereof, and the resultant is re-extracted with butanol.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
prevention and treatment of cardiovascular diseases, which
comprises an extract of Lindera obtusiloba, and health functional
food products. More particularly, the invention is characterized by
containing, as an active ingredient, an extract of Lindera
obtusiloba which has an effect of treating and preventing
cardiovascular diseases by directly inhibiting NAD(P)H oxidases to
suppress oxidative stress, and at the same time, activating
endothelial-type nitric oxide synthase to thereby regulate the
contraction and relaxation of vascular smooth muscle cells.
BACKGROUND ART
[0002] According to the statistics of the World Health Organization
(WHO), it is reported that the world death rate for cardiovascular
diseases in 1999 was 30% or greater, and the cardiovascular
diseases will be the first ranking diseases causing death in
developed countries in year 2010, while the increase rate of the
diseases is high particularly in Asian countries including Japan,
South Korea and the like. This is speculated to be attributable to
an increase in the risk factors for coronary artery diseases, as a
result of the coming aged society, changes in the dietary habits,
and the like.
[0003] Endothelial dysfunction is the main mechanism which induces
an extensive range of cardiovascular diseases, including
hypertension, arteriosclerosis, hyperlipidemia, diabetes mellitus,
obesity and the like (Brunner H. et al., J. Hypertens., 2005,
23:233-246), since the discovery of abnormal relaxation of blood
vessels in hypertensive patients in 1990 (Panza J A et al., New
England Journal of Medicine, 323:22-27, 1990). Endothelial cells
are epithelial cells lining along the cavities of blood vessels and
lymph vessels, and their main function is to produce vasodilators
and vasodilator mediators to regulate the vascular tone as well as
the structure.
[0004] A cardiovascular disease is a disease starting in the form
of initial endothelial dysfunction and finally resulting in
abnormality in the heart and vascular system. The name is a generic
term for a group of abnormalities in the heart and blood vessels,
including, but not limited to, arteriosclerosis, hypertension,
hyperlipidemia, coronary artery diseases (heart attack),
cerebrovascular diseases (cerebral stroke, dementia), peripheral
vascular diseases, arrhythmia, cardiac failure, congestive heart
diseases, myocardial diseases, and the like.
[0005] As for principal factors for the manifestation of
cardiovascular diseases, there are known a variety of factors such
as genetic factors, lifestyle, and complications of diabetes
mellitus. From the standpoint of modern medicine, increases in the
reactive oxygen species (ROS) and vascular oxidative stress as a
result of an increase in the activity of NADPH oxidases, and a
decrease in nitric oxide due to a decrease in the activity of
endothelial-type nitric oxide synthase (eNOS), are known as
principal factors. Nitric oxide which is produced by
endothelia-type nitric oxide synthase, is a potent vasorelaxant
factor, and also plays a critical role in the overall homeostatic
regulation of the cardiovascular system by inhibiting platelet
aggregation, proliferation of vascular muscle cells, vascular
adhesion of monocytes, and expression of
arteriosclerosis-associated proteins (Forstermann et al.,
Circulation, 113:1708-1714, 2006). However, an increase, caused by
a number of factors, in the activity of NADPH oxidases which are in
charge of the generation of active oxygen species in the blood
vessels, leads to a decrease in the production of nitric oxide
[Gryglewski et al., Nature, 320:454-456, 1986; Paravicini et al.,
Circulation Research, 91:54-61, 2002; Dusting et al., Clinical and
Experimental Pharmacology and Physiology, 25:S34-41, 1998], and the
active oxygen species thus produced bring about regulation of the
expression of adhesive molecules [Lo et al., Am. J. Physiol.,
264:L406-412, 1993], stimulation of the proliferation and migration
of vascular smooth muscle cells (VSMC) [Griendling and Ushio-Fukai,
J. Lab. Clin. Med., 132:9-15, 1998], regulation of lipoproteins
having oxidizing power, and the like, to thereby cause
cardiovascular diseases [Lynch and Frei, J. Lipid Res.,
34:1745-1753, 1993]. Furthermore, the increased generation of
active oxygen species in the blood vessels due to NAD(P)H oxidases,
is associated with the impaired function of endothelial nitric
oxide (NO) in patients having clinical risk factors for
atherosclerosis and coronary artery diseases. Fundamentally, the
generation of active oxygen species brings about contraction of
blood vessels [Guzik et al., Cir. Res., 86:E85-90, 2000]. In
general, reduction of active oxygen species, suppression of
downstream generation of active oxygen species, and induction of
the activity of endothelial-type nitric oxide synthase, all through
direct inhibition of NAD(P)H oxidases, are recognized as very
important targets in the prevention and treatment of cardiovascular
diseases [Forstermann et al., Circulation, 113:1708-1714, 2006;
Doggrell S A, Drug News Perspect., 17(9) 615-632, 2004; Inoguchi
T., Curr. Drug Targets, 6(4):495-501, 2005; Muzaffar S. et al.,
Trends Cardiovasc Med., 15(8):278-282].
[0006] To date, known as the substances inhibiting NAD(P)H oxidases
are diphenylene iodonium (DPI) and 4-hydroxy-3-methoxyacetophenone
(Apocynin) [Holland J, et al., U.S. Pat. No. 5,902,831]. However,
these substances have not been put into commercialization or
clinical use because of the problems of toxicity and specificity.
Furthermore, as nitric oxide regulators, there are available
nitroprusside and nitroglycerine. However, these substances are
also put into use only in the cases of emergency such as heart
attack, because of the problems of resistance and toxicity in
clinical uses.
[0007] Meanwhile, Lindera obtusiloba is a deciduous shrub of family
Lauraceae. It grows to a height of about 2 to 3 m, and the
flowering season is from March to April, while the fruiting season
is September. The fruits are pressed to obtain oil, which has been
used as a hair oil for women. The plant is called "Ginger Tree"
because the plant smells like ginger when the leaves or twigs are
plucked off, and is also known as Fool's Camellia or Japanese
spicebush. The plant is known to be efficacious in activation of
blood, slow twitching of muscle, anti-abscess and the like, and to
treat bruises, and swelling and pain due to blood extravasation,
and the plant is usually squashed and adhered to the site of wound.
The plant is mainly used against stomach ache, and is also used as
an antifebrile or a cough medicine. In private practices, leaves
and buds of the plant are brewed and taken like tea, as an
antifebrile or a cough medicine [Illustrated Book of Korean Plants,
by Chang-Bok Lee, 1980; Illustrated Book of Korean Folk Medicines,
by Jong-Hee Park, 2005, Encyclopedia of Local Medicines].
[0008] In regard to researches on the components of Lindera
Obtusiloba, Park, Jong-Chul, et al. (J. Korean Soc. Food Nutr.
1996. 25(1), p. 76-79) isolated quercitrin from the leaves, and
hyperoside from the twigs, while Kwon, Hak-Cheol, et al. (Archives
of Pharmacal Research, 22, p. 417-422, 1999) isolated actifolin,
pluviatiolol, 5,6-dihydromatairesinol, (+)-syringaresinol,
9-O-trans-feruloyl-5,5-dimethoxylariciresinol and the like. The
efficacies of the respective isolated components are reported to
include cancinocidal action, anti-inflammatory action, and the like
(Planta Medica, 69, 610-616, 2003; Archives of Pharmacal Research,
22, p. 417-422, 1999).
[0009] However, no disclosure or teaching is given in the
above-mentioned literatures, on that an extract of Lindera
obtusiloba has effects of suppression of vascular oxidative stress,
relaxation of blood vessels and regulation of blood pressure,
through inhibition of NADPH oxidases.
DISCLOSURE OF THE INVENTION
Technical Problem
[0010] Thus, the inventors of the present invention conducted an
investigation on the inhibition of NAD(P)H oxidase activity and
vasorelaxant effect with a number of plant extracts, and finally
confirmed the effects of potent inhibition of the activity of
NAD(P)H oxidases, reduction of vascular oxidative stress, direct
enhancement of the activity of endothelial type nitric oxide
synthase, relaxation of blood vessels and regulation of blood
pressure, thus completing the present invention.
[0011] The present invention was designed to solve the problems
such as described above, and an object of the invention is to
provide a composition for prevention and treatment of a
cardiovascular disease, comprising an extract of Lindera obtusiloba
as an active ingredient.
[0012] Another object of the present invention is to provide a
health functional food product for prevention of a cardiovascular
disease, comprising an extract of Lindera obtusiloba as an active
ingredient.
Technical Solution
[0013] To achieve the objects as described above, the composition
for prevention and treatment of a cardiovascular disease of the
present invention is characterized by containing an extract of
Lindera obtusiloba as an active ingredient.
[0014] The extract of Lindera obtusiloba is preferably an extract
obtained from the twigs, the leaves, or the twigs and leaves.
[0015] The extract of Lindera obtusiloba is preferably extracted
with a solvent selected from the group of water, a C1-C5 lower
alcohol, and a mixture thereof.
[0016] The extract of Lindera obtusiloba is preferably extracted
with a 30 to 95 wt % aqueous solution of ethanol.
[0017] It is preferable that the extract of Lindera obtusiloba be
extracted with a solvent selected from the group of water, a C1-C5
lower alcohol and a mixture thereof, and the extract be
re-extracted with butanol.
[0018] The extract of Lindera obtusiloba is characterized by having
an NAD(P)H oxidase inhibitory activity.
[0019] The extract of Lindera obtusiloba is characterized by having
a vasorelaxant effect.
[0020] The extract of Lindera obtusiloba is characterized by an
effect of enhancing the activity of endothelial-type nitric oxide
synthase.
[0021] The extract of Lindera obtusiloba is characterized by having
effects of decreasing the blood pressure and reducing the vascular
oxidative stress.
[0022] The cardiovascular disease may be selected from the group
consisting of congestive heart diseases, coronary artery diseases
(heart attack), ischemic heart diseases (myocardial ischemia),
hyperlipidemia, arteriosclerosis, hypertension, hypotension,
arrhythmia, cardiac failure, vascular restenosis, cerebrovascular
diseases (cerebral stroke, dementia), peripheral vascular diseases
and metabolic diseases.
[0023] Meanwhile, the health functional food product for prevention
of a cardiovascular disease of the present invention is
characterized by containing an extract of Lindera obtusiloba as an
active ingredient.
Advantageous Effects
[0024] As discussed in the above, the extract of Lindera obtusiloba
of the present invention strongly inhibits the activity of NAD(P)H
oxidases and exhibits a vasorelaxant effect at the same time, and
thus can be usefully utilized as a pharmaceutical composition or
health functional food product for prevention and treatment of a
cardiovascular disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a diagram showing the measurement of the
vasorelaxant activity of an extract of Lindera obtusiloba on swine
coronary arteries.
[0026] FIG. 2 is a diagram showing the measurement of the
vasorelaxant activity of an extract of Lindera obtusiloba on the
aorta of a white rat.
[0027] FIG. 3 is a diagram showing the measurement of the degree of
activity of endothelial-type nitric oxide synthase (eNOS) and Akt
protein under the action of an extract of Lindera obtusiloba.
[0028] FIG. 4 is a diagram showing the measurement of the degree of
influence of an extract of Lindera obtusiloba on the heart
rate.
[0029] FIG. 5 is a diagram showing the measurement of the degree of
decrease in vascular oxidative stress under the action of an
extract of Lindera obtusiloba.
[0030] FIG. 6 is a diagram showing the measurement of the degree of
cytotoxicity of an extract of Lindera obtusiloba.
BEST MODE FOR CARRYING OUT THE INVENTION
[0031] Hereinafter, preferred embodiments of the present invention
will be described in detail. In the following description, numerous
characteristic terms such as specific constituent elements are
illustrated, but these are provided only for the purpose of
providing an aid for more comprehensive understand of the present
invention, and it will be obvious to those having ordinary skill in
this technical field that the present invention can be carried out
even without these characteristic terms. Furthermore, in the
description of the present invention, if it is judged that specific
explanation of any related known function or constitution may
unnecessarily make the gist of the invention unclear, the detailed
explanation will be omitted.
[0032] The extract of Lindera obtusiloba of the present invention
can be obtained as follows.
[0033] The aerial part, more specifically, the leaves and twigs, of
Lindera obtusiloba can be used without limitation, in the form of
plants collected from nature, cultivated plants, commercially
available plants, or the like. The solvent for extraction is
selected from the group consisting of water, a C1-C5 lower alcohol,
and a mixture thereof. The inventors of the present invention
washed the twigs and leaves of Lindera obtusiloba to remove
impurities and salts, and dried them. Subsequently, extraction was
performed with a polar solvent such as water or a C1-C5 lower
alcohol such as methanol, ethanol or butanol, or a mixed solvent of
these at a mixing ratio of about 1:0.1 to 1:10, preferably with a
to 95 wt % aqueous solution of ethanol, in a volume reaching about
5- to 50-fold, and preferably 10- to 30-fold, the weight of the
Lindera obtusiloba sample, at 50 to 95.quadrature.C for 1 hour to 7
days. The above-described extraction process is repeated two or
five times, and preferably three times, and then the resultant is
subjected to concentration under reduced pressure and/or
freeze-drying, to obtain a crude extract of Lindera obtusiloba.
[0034] Among the extracts of the present invention, a non-polar
solvent-soluble extract can be obtained by suspending the
aforementioned crude extract in distilled water, subsequently
adding a non-polar solvent such as hexane, ethyl acetate or
chloroform in a volume of about 0.1 to 100 times, and preferably
about 1 to 5 times, the volume of the suspension, and performing
extraction and isolation once to 10 times, and preferably two to
five times. Furthermore, conventional fractionation processes can
also be additionally carried out (Harborne, J. B., Phytochemical
methods: A guide to modern techniques of plant analysis, 3rd Ed.,
pp. 6-7, 1998).
[0035] More preferably, the crude extract of Lindera obtusiloba
obtained by the above-described processes, preferably an extract of
Lindera obtusiloba in an aqueous solution of ethanol, is subjected
to sequential solvent fractionation with organic solvents such as
n-butanol, hexane and ethyl acetate, in order from less polar
solvent to more polar solvent, preferably in order of hexane, ethyl
acetate and n-butanol, and to concentration under reduced pressure,
and thus hexane, ethyl acetate and n-butanol fractions of Lindera
obtusiloba can be obtained.
[0036] The present invention provides a composition for prevention
and treatment of a cardiovascular disease, which comprises the
crude extract or non-polar solvent-soluble extract of Lindera
obtusiloba obtained by the above-described production method, as an
active ingredient.
[0037] The composition for prevention and treatment of a
cardiovascular disease according to the present invention contains
0.1 to 99% by weight of the aforementioned extract based on the
total weight of the composition.
[0038] The composition containing the extract of Lindera obtusiloba
of the present invention may further contain appropriate carriers,
excipients and diluents that are conventionally used in the
production of compositions.
[0039] The pharmaceutical dosage form of the extract of the present
invention can be used in the form of pharmaceutically acceptable
salts thereof, and also can be used alone, or in the form of
conjugate as well as appropriate assembly with other
pharmaceutically active compounds.
[0040] The pharmaceutical composition containing the extract
according to the present invention can be used after being
formulated into oral formulations such as powders, granules,
tablets, capsules, suspensions, emulsions, syrups and aerosols, and
into topical formulations, suppositories and sterile injectable
solutions, respectively according to conventional methods.
[0041] As for the carriers, excipients and diluents that can be
contained in the composition containing the extract, there may be
mentioned lactose, dextrose, sucrose, sorbitol, mannitol, xylytol,
erythritol, maltitol, starch, acacia gum, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methylcellulose,
non-crystalline cellulose, polyvinylpyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate, and mineral oil.
[0042] In the case of formulating into preparations, they are
prepared using conventionally used diluents or excipients, such as
fillers, bulking agents, binding agents, wetting agents,
disintegrants and surfactants.
[0043] Examples of solid preparations for oral administration
include tablets, pills, powders, granules, capsules and the like,
and these solid preparations are prepared by mixing at least one
excipient, for example, starch, calcium carbonate, sucrose or
lactose, gelatin and the like to the aforementioned extract.
Furthermore, in addition to simple excipients, lubricants such as
magnesium stearate and talc are also used.
[0044] Examples of liquid preparations for oral administration
include suspensions, liquids for internal use, emulsions, syrups
and the like. These liquid preparations may include various
excipients, for example, wetting agents, sweeteners, fragrances,
preservatives and the like, in addition to water and liquid
paraffins, which are frequently used simple diluents.
[0045] Examples of preparations for parenteral administration
include sterilized aqueous solutions, non-aqueous solutions,
suspensions, emulsions, freeze-dried preparations, and
suppositories. As non-aqueous solvents or suspending agents,
propylene glycol, polyethylene glycol, plant oils such as olive
oil, injectable esters such as ethyl oleate, and the like can be
used. As the base of the suppositories, witepsol, Macrogol, Tween,
cacao butter, laurin fat, glycerogelatin and the like can be
used.
[0046] A preferable amount of administration of the extract of the
present invention may vary with the condition and weight of the
patient, severity of the disease, form of drug, the route and
period of administration, but can be appropriately selected by a
person having ordinary skill in the art. However, for preferable
effects, it is desirable to administer the extract of the present
invention in an amount of 0.0001 to 100 mg/kg, and preferably 0.001
to 100 mg/kg, per day. Administration may be carried out once a
day, or may be carried out several times a day. The amount of
administration is not intended to limit the scope of the invention
by any means.
[0047] The present invention provides a health functional food
product containing the aforementioned extract showing an effect of
preventing a cardiovascular disease, and sitologically acceptable
food additives.
[0048] As for the health functional food products which can be
added with an extract of Lindera obtusiloba, for example, there may
be mentioned various general food products, beverages, chewing
gums, tea, vitamin complexes, and the like.
[0049] The extract of Lindera obtusiloba can also be added to foods
or beverages for the purpose of obtaining an effect of preventing a
cardiovascular disease. In this case, the amount of the extract in
a food product or beverage may be 0.01 to 15% by weight based on
the total weight of the food. The amount of the extract in a health
beverage composition may be 0.02 to 5 g, and preferably 0.3 to 1 g,
relative to 100 g of the total weight of the beverage.
[0050] The health functional beverage composition of the present
invention is not particularly limited in containing other
components, in addition to containing the extract as an essential
component at the indicated proportions, and can contain additional
components such as various flavoring agents or natural
carbohydrates, as conventional beverages do. Examples of the
aforementioned natural carbohydrates include conventional
saccharides, such as monosaccharides, for example, glucose,
fructose and the like; disaccharides, for example, maltose, sucrose
and the like; and polysaccharides, for example, dextrin,
cyclodextrin and the like, and sugar alcohols such as xylytol,
sorbitol and erythritol. As the flavoring agents in addition to the
aforementioned agents, thaumatin, stevia extracts, for example,
levaudioside A, glycyrrhizin and the like; and synthetic flavoring
agents, for example, saccharin, aspartame and the like can be
advantageously used. The proportion of the natural carbohydrates is
generally about 1 to 20 g, and preferably about 5 to 12 g, per 100
g of the composition of the present invention.
[0051] In addition to these, the extract of the present invention
may contain various nutrients, vitamins, minerals (electrolytes),
synthetic and natural flavoring agents, colorants and weighting
agents (cheese, chocolate and the like), pectic acid and salts
thereof, alginic acid and salts thereof, organic acids, protective
colloidal thickeners, pH adjusting agents, stabilizers,
antiseptics, glycerin, alcohols, carbonating agents used in
carbonated drinks, and the like. In addition thereto, the extracts
of the present invention may contain natural fruit juices, and
fruit fleshes for the production of fruit juice beverages and
vegetable beverages. These components can be used individually or
in combinations. In this case, the proportion of the additives is
not critical, but is generally selected from the range of 0.01 to
about 20 parts by weight per 100 parts by weight of the extract of
the present invention.
[0052] Hereinafter, the present invention will be described more
specifically, by making reference to the following Examples and
Test Examples.
Examples
Example 1
Preparation of Crude Extract of Lindera obtusiloba
[0053] The twigs and leaves of Lindera obtusiloba collected in
Kangwon-do Province were washed with water to remove impurities and
salts, and then were dried and crushed. In an extraction vessel, 25
g each of the crushed twigs or the crushed leaves of Lindera
obtusiloba, and 500 ml in total of a 70 wt % aqueous solution of
ethanol were added, and thermal extraction was repeatedly performed
three times at 70.quadrature.C, each time taking 3 hours, under
reflux cooling. The resultant was filtered through a filter paper,
and the filtrate was concentrated under reduced pressure in a water
bath at 40.quadrature.C, and was freeze-dried. As a result, 6.4 g
of a crude extract of the twigs of Lindera obtusiloba and 7.0 g of
a crude extract of the leaves of Lindera obtusiloba were
obtained.
Example 2
Preparation of Fractions of Crude Extracts of Lindera
obtusiloba
[0054] 5 g each of the crude extracts of the leaves and twigs of
Lindera obtusiloba obtained in Example 1 were respectively
suspended in 50 ml of purified water. The resultant was subjected
to sequential solvent fractionation three times respectively, with
50 ml each of hexane, ethyl acetate and n-butanol in this order, to
obtain the respective solvent fractions, and these were
concentrated under reduced pressure. As a result, a hexane
fraction, an ethyl acetate fraction and an n-butanol fraction of
Lindera obtusiloba were obtained.
Test Example 1
Measurement of Inhibitory Effect of Extracts of Lindera obtusiloba
on NAD(P)H Oxidase Activity
[0055] The crude extracts and fractions of Lindera obtusiloba
prepared in Example 1 and Example 2 as described above were
subjected to the measurement of an effect of inhibiting the
activity of NAD(P)H oxidases, which are index enzymes for the
development of cardiovascular diseases. To this end, the arterial
smooth muscle cells of white rat (Rat aortic smooth muscle cells;
RASMC) and the vascular endothelial cells of calf (Bovine aortic
endothelial cells; BAECs) were used to compare the changes in the
NADPH oxidase activity, and the results are as shown in the
following Table 1. First, the arterial smooth muscle cells and the
vascular endothelial cells were respectively mixed with MEM
(minimum essential medium), DMEM (Dulbecco's minimum essential
medium) and a 10% FBS (fetal bovine serum) solution, and the cells
were respectively incubated on a 96-well plate for 24 hours under
the conditions of 5% CO2/37.quadrature.C. Then, the cells were
incubated again for 24 hours in the culture solution with FBS
excluded. After the cells were stabilized, the cells were washed
with HBSS (Hank's balanced salt solution). These cells were shaken
with the leaf extract or twig extract of Lindera obtusiloba
obtained in the Example 1, and then the mixture was allowed to
react for 15 minutes and washed again with HBSS. Subsequently, 100
mM NAD(P)H and 5 .quadrature.M Lucigenin were added to the reaction
liquid, and the activity of NAD(P)H oxidases was measured for one
hour, using a luminescence reader (Victor Light, PerkinElmer). The
test was operated three times, while repeated 2 to 7 times for each
operation. Subsequently, the activity of the control group and the
activity of the sample-treated group were compared, and the 1050
values were calculated. The 1050 value is the concentration,
expressed in .quadrature.g/ml, of a test material, at which 50% of
the NADPH oxidase activity is inhibited.
TABLE-US-00001 TABLE 1 Inhibitory effect of extracts of Lindera
obtusiloba on NADPH oxidase activity (unit: .mu.g/ml) IC50 IC50
(BAECs) (RASMC) Crude twig extract of 2.1 2.0 Lindera obtusiloba
Butanol fraction of twigs of 0.9 1.5 Lindera obtusiloba Ethyl
acetate fraction of 1.0 2.5 twigs of Lindera obtusiloba Hexane
fraction of twigs of 10.5 >30 Lindera obtusiloba
[0056] As shown in the Table 1, it can be seen that the extracts of
Lindera obtusiloba of the present invention are excellent in the
extent of inhibiting the activity of NADPH oxidases of the vascular
cells. Furthermore, among the fractions, it can be seen that the
n-butanol extract shows excellent effects.
Test Example 2
Measurement of Vasorelaxant Effect of Extracts of Lindera
obtusiloba (Coronary Arteries and Aorta)
[0057] The vasorelaxant effects of the crude extracts and fractions
of Lindera obtusiloba prepared in the Example 1 and Example 2 were
verified. To this end, a comparison was made on the effects of
relaxing the coronary arteries of a pig heart and the rat aorta,
and the results are as shown in FIG. 1, FIG. 2, Table 2 (coronary
arteries) and Table 3 (aorta). The coronary arteries were extracted
from a pig heart purchased immediately after slaughter at the
butchery. The aorta was extracted from a male SD (Sprague-Dawley)
white rat, was immersed in the Krebs solution (pH 7.4) containing
18 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4,
1.5 mM CaCl.sub.2, 25 mM NaHCO.sub.3, and 10 mM glucose to remove
the connective tissue and fats, and then was cut into a specimen of
about 3 mm in length.
[0058] The prepared coronary artery and aorta specimen were fixed
in the Krebs solution at 37.degree. C., which had been saturated
with 95% O.sub.2 and 5% CO.sub.2 gases. The isometric tensions of
the specimens were measured with a Grass physiograph (Hugo Sachs,
Germany) equipped with a force-displacement transducer (Hugo Sachs,
Germany). In the case of the coronary arteries, the arteries were
first contracted to 80% of the maximum vascular contraction by
using U46619 (1 to 60 nM), which is a thromboxane derivative. After
10 minutes therefrom, the arteries were relaxed with 300 nM
Bradykinin, the safety of the vascular endothelial cells was
measured, and then the arteries were washed three times with the
Krebs solution to perform the experiment. The measurement of
changes in the vasorelaxant effect was carried out by first
contracting the arteries with the drug U46619, and then the
relaxation reaction induced by the extracts of Lindera obtusiloba
was tested in a concentration-dependent manner.
[0059] In the case of the aorta of white rat, the aorta was
contracted to 80% of the maximum vascular contraction by using
phenylephrine. After 15 minutes therefrom, the aorta was relaxed
with 10 .mu.M acetylcholine, the safety of the vascular endothelial
cells was measured, and then the aorta was washed three times with
the Krebs solution to perform the experiment. The measurement of
changes in the vasorelaxant effect was carried out by first
contracting the aorta with the drug phenylephrine, and then the
relaxation reaction induced by the crude extracts and fraction
extracts of Lindera obtusiloba was tested in a
concentration-dependent manner. This test was repeatedly carried
out using the blood vessels extracted from 5 to 10 different
individuals. The ED50 value refers to the concentration (.mu.g/ml)
of the sample at which the contracted blood vessels show 50%
vasorelaxation as a result of the treatment with the sample.
TABLE-US-00002 TABLE 2 Relaxant effect of extracts of Lindera
obtusiloba on swine coronary arteries (unit: .mu.g/ml) ED50 Crude
leaf extract of Lindera 59.4 obtusiloba Crude twig extract of
Lindera 12.9 obtusiloba Butanol fraction of leaves of 18.6 Lindera
obtusiloba Ethyl acetate fraction of >100 leaves of Lindera
obtusiloba Hexane fraction of leaves of >100 Lindera obtusiloba
Butanol fraction of twigs of 6.4 Lindera obtusiloba Ethyl acetate
fraction of 71.1 twigs of Lindera obtusiloba
TABLE-US-00003 TABLE 3 Relaxant effect of extracts of Lindera
obtusiloba on white rat aorta (unit: .mu.g/ml) ED50 Crude leaf
extract of Lindera 14.8 obtusiloba Crude twig extract of Lindera
5.0 obtusiloba Butanol fraction of leaves of 5.4 Lindera obtusiloba
Ethyl acetate fraction of 48.3 leaves of Lindera obtusiloba Hexane
fraction of leaves of 79.6 Lindera obtusiloba Butanol fraction of
twigs of 1.7 Lindera obtusiloba Ethyl acetate fraction of 15.0
twigs of Lindera obtusiloba Hexane fraction of twigs of 95.0
Lindera obtusiloba
[0060] As a result of the test, as shown in FIG. 1 and FIG. 2, the
extracts of Lindera obtusiloba of the present invention started
significant relaxation from the concentration of 1 to 10 .mu.g/ml,
and reached 94.+-.2% relaxation at the concentration of 30
.mu.g/ml. As shown in Table 1 and Table 2, it can be seen that the
extracts of Lindera obtusiloba are excellent in the extent of
relaxing the blood vessels of the coronary arteries and the aorta.
Among the fractions, it can be seen that the n-butanol extract
shows excellent effects.
Test Example 3
Test with Extracts of Lindera obtusiloba on Endothelial Type Nitric
Oxide Synthase Activity
[0061] Observations were made on the effects of the crude extracts
of Lindera obtusiloba prepared in the Example 1 and Example 2 on
the enhancement of the activity of endothelial type nitric oxide
synthase. The above-mentioned effects were compared on the basis of
the degree of phosphorylation of serine residue 1177 of the
endothelial type nitric oxide synthase (eNOS) and serine residue
473 of Akt of bovine aortic endothelial cells. The results are
presented in FIG. 3.
[0062] First, the vascular endothelial cells were mixed with DMEM
(Dulbecco's minimum essential medium) and a 10% FBS (Fetal bovine
serum) solution and incubated, and then the cells were incubated
again for 24 hours in the culture solution with FBS excluded. After
the cells were stabilized, the cells were treated with the samples
at the respective concentrations, and then were allowed to react
for 30 minutes. Then, the proteins were extracted and centrifuged,
and the supernatant was collected to remove debris of the cells.
The extracted proteins were subjected to electrophoresis on
SDS-polyacrylamide gel, and then the proteins in the gel were
blotted with a nitrocellulose membrane. After blocking with 3% BSA
for one hour, the antibodies of phospho-eNOS and phospho-Akt (Cell
Signaling, the USA) were incubated overnight at 4.degree. C. at a
ratio of 1:1,000. Subsequently, secondary antibodies diluted at a
ratio of 1:2000 were treated and incubated at ambient temperature
for one hour, and then the secondary antibodies were developed by
chemiluminescence.
[0063] As a result, as shown in FIG. 3, it can be seen that the
extracts of Lindera obtusiloba show high effects of enhancing the
activation of endothelial type nitric oxide synthase
(phospho-eNOS), which produces nitric oxide, and a high degree of
increase in the activation of Akt (phospho-Akt), which is an
upstream regulatory factor.
Test Example 4
Test of Effects of Extracts of Lindera obtusiloba in Animal Model
of Cardiovascular Disease
[0064] The hypotensive effects in diseased animals, and the effects
on the improvement of vascular oxidative stress and the improvement
of endothelial dysfunction, exerted by the extracts of Lindera
obtusiloba obtained in Example 2, were verified. To this end, a
comparison was made on the extent of the effects in a disease model
induced by angiotensin 2, and the results are presented in FIG. 4,
FIG. 5, FIG. 6 and Table 4.
[0065] Male SD (Sprague-Dawley, 6 weeks old) white rats were
purchased from Orientbio, Inc. and the rats were acclimatized for
one week while freely supplying solid feedstuff and water in a
small animal breeding chamber which was regulated to have a
light-dark period of 12 hours. Then, the rats were arbitrarily
grouped into a control group and an angiotensin 2-treated group,
with each group including 6 animals. From three days before the
treatment with angiotensin 2, the extracts of Lindera obtusiloba
obtained in Example 2 were respectively suspended in 0.5% CMC
(carboxymethylcellulose) at a concentration of 100 mg/kg, and the
suspensions were orally administered twice a day. The control group
was administered with 0.5% CMC only. Angiotensin 2 was dissolved in
physiological saline at a concentration of 65 ng/min/kg, and the
solution was placed in a mini-osmotic pump (Alzet Model 2002). The
white rats were anesthetized, and the angiotensin 2 treatment was
carried out by cutting the skin open, and inserting the mini-pump
in the interscapular region. The blood pressure measurement was
conducted simultaneously with the initiation of oral
administration, and further measurements were made once in two days
for 2 weeks, one hour after the oral administration in the morning.
The specimen was heated in advance for about 10 minutes at 45 to
50.degree. C., and then the maximum blood pressure (systolic
pressure) of the tail artery was measured in a non-invasive manner
by the tail-cuff plethysmography method using an automated blood
pressure recording system. The heart rate was also measured at the
same time.
[0066] The aorta was extracted in the same manner as in Test
Example 2, and the degree of staining by DHE (dihydroethidium) and
the degree of relaxation by acetylcholine were characterized to
measure the extents of vascular oxidative stress and vascular
endothelial dysfunction. The results were compared with the results
of the control group.
TABLE-US-00004 TABLE 4 Hypotensive effects of extracts of Lindera
obtusiloba (unit: mmHg) 1.sup.st day 7.sup.th day 14.sup.th day
Control 107.6 107.4 112.6 Angiotensin 2 110.3 169.6 158.3 Twig
extract of 113.9 129.7 127.4 Lindera obtusiloba Leaf extract of
112.5 144.2 145.4 Lindera obtusiloba
[0067] As a result of the test, it can be seen from the Table that
the blood pressure increase in the test animals induced by
angiotensin 2 was significantly inhibited by the administration of
the extracts of Lindera obtusiloba.
[0068] The increase in the heart rate was measured, and as a
result, as shown in FIG. 4, it was confirmed that the extracts of
Lindera obtusiloba do not have adverse side effects on the heart,
such as tachycardia.
[0069] Furthermore, as shown in FIG. 5, it can be seen that the
increase in vascular oxidative stress induced by angiotensin 2 is
strongly inhibited by the administration of the extracts of Lindera
obtusiloba (FIG. 5 is a product of black-and-white modification of
a photograph which indicates the object in red and the background
in black, and at the time of modification, the background was
rendered white for clear distinction of the object).
[0070] The relaxation induced by 0.01 .mu.M acetylcholine in the
animals treated with angiotensin 2, was compared with that in the
control group, and it was found that vascular endothelial
dysfunction occurred in which relaxation does not occur smoothly
(control group--27%, acetylcholine group--8%). It can be seen that
such vascular endothelial dysfunction is improved by the
administration of the twig extract (20%) and leaf extract (19%) of
Lindera obtusiloba.
Test Example 5
Test on Cytotoxicity of Extracts of Lindera obtusiloba
[0071] A comparison was made for the crude extracts of Lindera
obtusiloba prepared in the Example 1 and Example 2 on the extent of
cytotoxicity, and the results are as shown in FIG. 6.
[0072] Arterial smooth muscle cells were mixed with MEM (minimum
essential medium) and a 10% FBS (fetal bovine serum) solution, and
the cells were incubated for 24 hours under the conditions of 5%
CO.sub.2/37.degree. C. After the cells were stabilized, the cells
were treated with the extracts of Lindera obtusiloba obtained in
the Example 2, shaken, and allowed to react for 24 hours.
Thereafter, the cells were incubated for one hour in the presence
of an MTS solution (CellTiter 96 Aqueous One Solution, Promega),
and then the absorbance at 490 nm was measured.
[0073] As shown in FIG. 6, it can be seen that the extracts of
Lindera obtusiloba of the present invention were not found to have
any influence on the survival of cells, and are very safe
drugs.
Test Example 6
Statistical Processing
[0074] The significance of the test results was determined by
performing the Student t-test and one-way ANOVA test with the
experimental results, such that there is a significant difference
in the case where p is 0.05 or less.
[0075] Preparation examples of the pharmaceutical composition and
health functional food products containing the extract of the
present invention will be described in the following, but the
present invention is intended not to limit them, but to
specifically describe them.
Preparation Example 1
Production of Powder Preparation
[0076] Powdered extract of Lindera obtusiloba 20 mg [0077] Lactose
100 mg [0078] Talc 10 mg
[0079] The components were mixed and filled in a sealed pack, and
thereby a powder preparation was produced.
Preparation Example 2
Production of Tablet Preparation
[0080] Powdered extract of Lindera obtusiloba 10 mg [0081] Corn
starch 100 mg [0082] Lactose 100 mg [0083] Magnesium stearate 2
mg
[0084] The components were mixed and then tableted according to a
conventional tablet production method, and thereby a tablet
preparation was produced.
Preparation Example 3
Production of capsule Preparation
[0085] Powdered extract of Lindera obtusiloba 10 mg [0086]
Crystalline cellulose 3 mg [0087] Lactose 14.8 mg [0088] Magnesium
stearate 2 mg
[0089] According to a conventional capsule production method, the
components were mixed and filled in gelatin capsules, and thereby a
capsule preparation was produced.
Preparation Example 4
Production of Injectable Preparation
[0090] Powdered extract of Lindera obtusiloba 10 mg [0091] Mannitol
180 mg [0092] Sterile distilled water for injection 2794 mg [0093]
Na.sub.2HPO.sub.412H.sub.2O 26 mg
[0094] According to a conventional production method for injectable
preparation, an injectable preparation was produced at the
aforementioned component contents per ampoule (2 ml).
Preparation Example 5
Production of Liquid Preparation
[0095] Powdered extract of Lindera obtusiloba 10 mg [0096]
High-fructose corn syrup 10 g [0097] Mannitol 5 g [0098] Purified
water Appropriate amount
[0099] According to a conventional production method for liquid
preparation, the components were added and dissolved in purified
water, an appropriate amount of lemon flavor was added thereto, the
components were mixed, and the total volume was adjusted to 100 ml
by adding purified water. This was filled in a brown bottle and
sterilized, and thereby a liquid preparation was produced.
Preparation Example 6
Production of Health Drink
[0100] Powdered extract of Lindera obtusiloba 10 mg [0101] Vitamin
C 15 g [0102] Vitamin E (powdered) 100 g [0103] Iron lactate 19.75
g [0104] Zinc oxide 3.5 g [0105] Nicotinic acid amide 3.5 g [0106]
Vitamin A 0.2 g [0107] Vitamin B1 0.25 g [0108] Vitamin B2 0.3 g
[0109] Water Appropriate amount
[0110] According to a conventional production method for health
drink, the components were mixed, the mixture was heated under
stirring at 85.degree. C. for about one hour, and the resulting
solution was filtered. The filtrate was placed in a sterilized
2-liter container, sealed, sterilized, and refrigerated, and this
was used in the production of the health drink compositions
according to the present invention. In the above-given
compositional ratios, components which are relatively suitable for
favorite drinks were mixed and composed as a preferred Example, but
the mixing ratio may be arbitrarily modified in accordance with the
geographical and racial preference, such as of the class of
consumers or consumer country, the uses, or the like.
* * * * *